In various industrial fields, researchers have studied to develop more sophisticated preservation technology for biological cells or tissues. For example, in bovine embryo transplantation technology, an embryo is transplanted corresponding to the estrous cycle of the recipient cattle of the embryo. In order to carry out the embryo transplantation, the embryo is generally cryopreserved and then is thawed for the transplantation corresponding to the estrous cycle. In the infertility treatment of human, after an ovum or ovary is collected from a mother, it is cryopreserved and then is thawed upon the transplantation to use.
Generally, even though in a culture solution, long term culture of a cell or tissue placed outside a living body is undesirable since the cell or tissue collected from the living body gradually loses its biological activity. Therefore, it is very important to develop technology for long-term preservation of the cell without losing its biological activity. The development of the technology is expected to allow the collected cell or tissue to be analyzed with high accuracy and used for transplantation while keeping its higher biological activity, which results in the improvement of engraftment rate after the transplantation. In addition, the development of the technology is also expected to allow a skin cultured in vitro or an artificial tissue constructed in vitro, such as what is called cell sheet, to be sequentially produced and preserved for use when necessary. Thus, the development of the technology is expected to bring us significant advantages in terms of industries as well as medical treatment.
As a preservation method for cell or tissue, for example, the slow freezing method has been known. The first step of this method is to immerse a cell or tissue in a preservation solution obtained by incorporating a cryoprotectant into a physiological solution such as phosphate buffered saline. As a cryoprotectant, some compounds such as glycerol, ethylene glycol are used. After immersing the cell or tissue in the preservative solution, cooling it to −30 to −35° C. at a relatively slow cooling rate (e.g., speed of 0.3 to 0.5° C./min.) provides a sufficient refrigeration and a high viscosity for the solutions inside and outside of the cell or tissue. In such a state, further cooling to the temperature of liquid nitrogen (−196° C.) for the cell or tissue in the preservation solution causes the vitrification phenomenon in which a very small amount of solutions existing inside of the cell or tissue and around outside thereof both turn solid while remaining non-crystalline. Since the solidification inside and outside the cell or tissue by vitrification makes the movement of the molecules therein substantially stop, the cell or tissue vitrified in liquid nitrogen would be preserved semi-permanently.
However, since the slow freezing method mentioned above requires cooling at a relatively low cooling rate, it takes time to carry out the cryopreservation. Also, there is a problem that the method requires a device or jig for controlling temperature. In addition, since the slow freezing method forms ice crystals in the preservation solution outside the cell or tissue, the cell or tissue may suffer physical damage from the ice crystals.
As a method for solving the problems in the slow freezing method, a vitrification preservation method has been proposed. The vitrification preservation method applies the principle that ice crystals are hardly produced even below zero by cryoscopy of a solution containing a large amount of cryoprotectant such as glycerol, ethylene glycol, or DMSO (dimethyl sulfoxide). Rapid refrigeration of this solution in liquid nitrogen can bring solidification while not making ice crystals. Such solidification is called vitrification congelation. Also, the solution containing a large amount of cryoprotectant is called vitrification solution.
A specific handling of the vitrification method is to immerse a cell or tissue in the vitrification solution and then to freeze it at the temperature of liquid nitrogen (−196° C.) Since the vitrification method is such a simple and rapid process, it has some advantages that there is no need to take time to carryout the cryopreservation, and no need for a device or jig for controlling temperature.
With the vitrification method, it is possible to avoid physical damage (frost damage) to the cell during freezing and during thawing for not producing ice crystals inside nor outside of the cell. On the other hand, the cryoprotectant of high concentration in the vitrification solution has chemical toxicity. Thus, it is preferable for the vitrification solution existing around the cell or tissue during cryopreservation to be less, and for the time when the cell is exposed to the vitrification solution, namely the time until the freezing, to be short. Furthermore, there is a need to dilute the vitrification solution immediately after thawing.
Regarding the cryopreservation of a cell or tissue using the vitrification preservation method, examples of various ways using various types of the cell or tissue are illustrated. For example, Patent Document 1 shows that the application of vitrification preservation method to a reproductive or somatic cell of an animal or human is extremely useful in terms of the survival rate after the cryopreservation and thawing.
The vitrification preservation method is a technology developed chiefly using the reproductive cell of human. Nowadays, the application to iPS cell or ES cell is widely investigated. Further, Non-Patent Document 1 shows that the vitrification preservation method was effective in the preservation of Drosophila embryo. Furthermore, Non-Patent Document 2 shows that the vitrification preservation method is effective in the preservation of a cultured plant cell and tissue. Thus, the vitrification method is known to be useful for the preservation of a wide variety of species of cell and tissue.
As a jig and an operation method for more efficiently performing the vitrification preservation method, Patent Document 2 and Patent Document 3, etc. illustrate an attempt that is made to improve the regeneration rate of ovum or embryo by performing the vitrification cryopreservation thereof in a straw filled with the vitrification solution and by being quickly contacted with a dilution solution at the time of thawing.
Patent document 4 describes a method that can cryopreserve the reproductive cell of ovum or embryo with high survivability by absorbing the extra vitrification solution attached around it with an absorber such as a filter paper.
Patent Documents 5 and 6 describe what is called Cryotop method, which is used in the infertility treatment field of human. The method uses a tool for ovum cryopreservation with a film of a strip for the ovum adhesion retention which film is flexible and colorless transparent, and makes the ovum or embryo adhere to the film together with a very small amount of the vitrification solution under the microscope to cryopreserve.
Patent Document 7 describes a method of cryopreserving a collected tissue fragment using a plate for tissue fragment cryopreservation, which plate is made of a metallic board-formed material, and has a lot of holes to which a cooling medium can gain entry.